Objective To determine the effect of different fluid shear stress on proliferation and osteogenic differentiation of human dermal fibroblasts in three-dimensional culture condition through a plane homogeneous flow field bioreactor. Methods Human dermal fibroblasts were pre-inoculated in collagen hydrogel scaffold with a density of 2×105/mL and divided into 3 groups treated with different shear stress. The low flow velocity treatment group (LFV group) was treated with fluid shear stress of 8 mPa, the high flow velocity treatment group (HFV group) was treated with fluid shear stress of 23 mPa, and the control group was treated without any shear stress. HE staining and cell counting were performed on days 3, 6 and 9. Glucose consumption in the medium was tested every 3 d. Alizarin Red S staining was used to measure mineralized nodule formation on day 21, and the expression of alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2) was determined by FQ-PCR. Results HE staining and cell counting assays showed that the cell proliferations were significant in all 3 groups, especially in the LFV group on days 6 and 9 (P<0.05). The glucose consumption of the 3 groups were all decreased along with time but showed no significant difference among them. On day 21, Alizarin red staining showed that the 3 groups were all positive, and the HFV group had the largest mineralized nodule area (P<0.05). The expression level of ALP and BMP-2 were 1.780±0.233 and 2.788±0.241 in the LFV group, 2.262±0.306 and 4.576±0.253 in the HFV group, and 1.147±0.215 and 0.354±0.063 in the control group, respectively (P<0.05). Conclusion Fluid shear stress promotes the proliferation and osteogenic differentiation of human dermal fibroblasts, and the cell proliferation is strengthened with smaller fluid shear stress and inhibited with larger shear stress.